CYTOGENETIC CHARACTERIZATION OF THE FLORIDA MANATEE (Trichechus manatus latirostris) BY CHROMOSOME BANDING TECHNIQUES
B Gray1, R Zori1, P McGuire2, S Felton1, R Bonde3
1Division of Genetics, College of Medicine, University of Florida;
2 Department of Biochemistry & Molecular Biology, University of Florida;
3 U.S. Department of the Interior, Florida Integrated Science Center, Florida.
Presented at the 14th Biennial Conference on the Biology of Marine Mammals,
28 November - 3 December 2001, Vancouver, BC, Canada.
Published cytogenetic data for the Order Sirenia is limited and, thus far, karyotypes produced have been restricted to conventional or solid chromosome staining techniques. To facilitate identification of individual chromosome homologs for the Florida manatee (Trichechus manatus latirostris), we have applied primary chromosome banding techniques, G- and Q-banding, to metaphase chromosomes prepared from T- and B-cell peripheral blood lymphocyte cell cultures established from six individuals (three males; three females). Following brightfield and fluorescence microscopic analyses, a previously published modal chromosome number of 48 was confirmed for this species. Digital imaging methods were subsequently employed and individual homologues were identified by unique G-band patterns and chromosome morphologies. A standard banded karyotype was constructed, for both sexes, based on the G-band chromosome pattern obtained in these studies. Characterization of additional cytogenetic features of this species by supplemental chromosome banding techniques, C-banding (constitutive heterochromatin), Ag-NOR staining (nucleolar organizer regions), and DA/DAPI staining was also performed. Cytogenetic features, including chromosome morphology and banding patterns, of Trichechus manatus latirostris are described. These studies may provide a basis for more precise inter and intra specific comparisons by cytogenetic methodologies.
Of the four species of the modern sirenians (manatees and dugongs), we know of only one, the Amazonian manatee (Trichechus inunguis), which has been studied by cytogenetic methods that included the use of chromosome banding techniques (Assis et al., 1988). Published karyotypic data for the remaining species, has been limited to the Florida manatee (Trichechus manatus latirostris) and the dugong (Dugong dugon). Unfortunately, the existing cytogenetic studies have been restricted to solid stained chromosomes, which prohibit the precise identification of individual chromosome homologs, chromosome regions, and/or chromosome bands.
A renewed interest in the chromosomal or cytogenetic status of various species has been generated by the advancements of genetic mapping techniques utilizing fluorescence in situ hybridization (FISH) techniques and related comparative genomic techniques such as Zoo-FISH. This type of work would undoubtedly benefit from the availability of a more detailed or precise karyotype for the species being studied.
We present the first karyotypic data generated by multiple chromosome banding techniques, including G-banding, Q-banding, C-banding, Ag-NOR staining, and DA/DAPI staining for the Florida manatee (Trichechus manatus latirostris). Combining the chromosome morphology and banding patterns, we were able to identify each homolog pair and consistently construct an accurate karyotype for six individuals.
MATERIALS AND METHODS
Blood Collection & Culture:
The medial aspect of the manatee's flipper was surgically scrubbed and sterilized. Venipuncture was accomplished through a sterile 18-20 gauge, 1.5 inch needle inserted into the palpable interosseous space between the radius and ulna. This blind stick includes target vessels within a small plexus (brachial vascular bundle) deep within the tissue. Vacutainers were used to draw approximately 20 mls of blood into green top sodium heparin tubes. Samples were placed on ice and transported to the laboratory as soon as possible for cytogenetic processing. Transit time ranged from 3 to 36 hours.
Buffy coats obtained from the blood were cultured at 36oC for 72 hours in RPMI medium (supplemented with 20% fetal bovine serum, 0.8% L-glutamine, and 0.4% penicillin-strepomycin) with 150µl of phytohemagglutinin (L & M-forms) or pokeweed. Mitotic cells were harvested following modifications of the procedure as described by Brown MG (1997).
Microscope slides were cleaned by dipping in methanol followed by a rinse in deionized water. The slides were covered with distilled water and stored at 4ºC until ready for use. The lymphocyte suspension was diluted with fix solution to achieve a slightly cloudy mix. Several drops of this suspension were placed on a cold and wet microscope slide and allowed to air dry. Metaphase spreading was assessed by phase microscopy and adjustments were made to accommodate drying times affecting spreading and residual cytoplasm.
G-banded chromosome preparations were obtained for all six individuals by modifications of the trypsin (GTG) and Giemsa staining procedure as described by Seabright, M (1971).
Q-banding was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Gustashaw, KM (1997).
C-banding was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Benn, PA and MA Perle (1986).
Ag-NOR staining was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Verma, RS and KA Babu(1984).
DA/DAPI staining was performed for two (one male and one female) of the six individuals following modifications of the procedure as described by Benn, PA and MA Perle (1986)
Imaging and Karyotype Production:
A minimum of three metaphase images and karyotypes per individual were prepared by computer digital imaging methods.